The present invention relates generally to a method for treating diseases associated with toxicity of Apolipoprotein E ("apoE"). Specifically, the present invention is a new method for treating a mammal having a condition associated with toxicity of apolipoprotein E cleavage fragments containing residues 130-169, comprising administering to said mammal a pharmacologically effective amount of compound or a pharmaceutically acceptable salt, derivative or fragment thereof to interfere with generation of the cleavage fragment or with the receptor-binding site associated with the cleavage fragment containing residues 130-169 of the apolipoprotein E molecule in said mammal.
Alzheimer's disease is the most common form of both senile and pre-senile dementia in the world and is recognized clinically as relentlessly progressive dementia that presents with increasing loss of memory, intellectual function and disturbances in speech (Merritt, 1979, A Textbook of Neurology,. 6th edition, pp. 484-489 Lea & Febiger, Philadelphia). The disease itself usually has a slow and insidious progress that affects both sexes equally, worldwide. It begins with mildly inappropriate behavior, uncritical statements, irritability, a tendency towards grandiosity, euphoria and deteriorating performance at work; it progresses through deterioration in operational judgment, loss of insight, depression and loss of recent memory; it ends in severe disorientation and confusion, apraxia of gait, generalized rigidity and incontinence (Gilroy & Meyer, 1979, Medical Neurology, pp.175-179 MacMillan Publishing Co.) Alzheimer's disease afflicts an estimated four million human beings in the United States alone at a cost of 35 billion dollars a year (Hay & Ernst, 1987, Am. J. Public Health, 77: 1169-1175). It is found in 10% of the population over the age of 65 and 47% of the population over the age of 85 (Evans et al., 1989, JAMA, 262:2551-2556). In addition, the disease is found at much lower levels in the younger age groups, usually beginning at about 30 years of age and even rarely in late childhood (Adams & Victor, 1977, Principles of Neurology, pp. 401-407).
The etiology of Alzheimer's disease is unknown. Evidence for a genetic contribution comes from several important observations such as the familial incidence, pedigree analysis, monozygotic and dizygotic twin studies and the association of the disease with Down's syndrome (for review see Baraitser, 1990, The Genetics of Neurological Disorders, 2nd edition, pp. 85-88). Nevertheless, this evidence is far from definitive and it is clear that one or more other factors are also required.
In recent years, research has suggested that apolipoprotein E ("apoE") plays a potential role in the pathogenesis of Alzheimer's disease. Apolipoprotein E performs various functions as a protein constituent of plasma lipoproteins, including its role in cholesterol metabolism. It was first identified as a constituent of liver-synthesized very low density lipoproteins ("VLDL") which function in the transport of triglycerides from the liver to peripheral tissues. ApoE is instrumental in lipoprotein metabolism in several ways. Mahley, et al., J. Lipid Res., 25:1277-1294 (1984). It is a recognition site for several cellular lipoprotein receptors, including hepatocyte receptors for chylomicron and VLDL remnants Hui, et al., J. Biol. Chem., 259:860-869 (1984); Shelburne, et al., J. Clin. Invest., 65:652-658 (1980).
ApoE-enriched lipoproteins have also been described to have a function in the immune system by inhibiting mitogen-or antigen-stimulated lymphocyte proliferation in vitro and in vivo. In the ovary, apoE inhibits androgen production by LH-stimulated cultured theca and interstitial cells; Dyer, et al., J. Biol. Chem., 263:10965 (1988).
Further substantiation that apoE and apoB-containing lipoproteins are important regulators of lymphocyte function has come from studies of the inhibitory properties of fetal cord blood plasma lipoproteins (Curtiss, et al., J. Immunol., 133:1379 (1984)). In these studies a direct correlation between apoE and inhibition was established.
There are three major isoforms of ApoE, referred to as ApoE2, ApoE3 and ApoE4 which are products of three alleles at a single gene locus. Three homozygous phenotypes (Apo-E2/2, E3/3, and E4/4) and three heterozygous phenotypes (ApoE3/2, E4/3 and E4/2) arise from the expression of any two of the three alleles. The most common phenotype is ApoE3/3 and the most common allele is E3. See Mahley, R. W., Science 240:622-630(1988).
The amino acid sequences of the three types differ only slightly. ApoE4 differs from ApoE3 in that in ApoE4 arginine is substituted for the normally occurring cysteine at amino acid residue 112. The most common form of ApoE2 differs from ApoE3 at residue 158, where cysteine is substituted for the normally occurring arginine. See Mahley, Science, supra. ApoE phenotypes and genotypes are well described and known in the art as described above. The established nomenclature system as well as the phenotypes and genotypes for ApoE, are described in, for example, Zannis, et al., J. Lipid. Res. 23:911 et seq. (1982), which is incorporated by reference herein.
Subjects with the ApoE4/4 genotype are as much as eight times as likely to be affected by Alzheimer's disease as subjects with the ApoE2/3 or ApoE3/3 genotypes. Further, the average age of onset of Alzheimer's disease and the average age of survival is lower for those having one ApoE4 allele, and lowest for those having two ApoE4 alleles. Thus, a subjects prognosis for Alzheimer's disease is more likely to be negative if the subject has an ApoE4 allele and most negative if the subject has more than one ApoE4 allele. The negative prognosis can be viewed in terms of increased likelihood of developing the disease, or of dying at an earlier age. Other ApoE-linked diseases include type III Hyperlipidemia and atherosclerosis.
Studies have shown that apoE fragments ranging from 5 to 22 kD are present in the post-mortem cerebral spinal fluid from both control patients and patients with AD. The only major band immunoprecipitated by a monoclonal antibody that recognizes the putative toxic domain runs with an apparent molecular weight of about 22 kD. This fragment likely corresponds to the major thrombin cleavage product, which has been shown to be protease-resistant. Weisgraber, et al., J. Biol. Chem. 258:12348-54 (1983).
Amino acids 130-169 in human apoE encompass an immunoregulatory domain with both cytostatic and cytotoxic activities against IL2-dependent T cells. This finding is consistent with results of previous studies (Cardin, et al., 1988; Dyer, et al., 1991) that implicated residues 141-155 in apoE's antiproliferative effect on naive mitogen-activated T cells. The similar potencies of E130-149 and E130-155 indicate that the cytostatic domain is located within residues 130-149. However, a longer peptide representing residues 130-169 and dimeric peptides of amino acids 141-155 also have potent cytotoxic activity. These results indicate that the positively charged, leucine-rich sequence, corresponding to amino acids 141-149 (Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu; referred to as LRKLRKRLL in single letter amino acid shorthand; SEQ. ID. No:1) in the mature protein which represents the overlap between the functional peptides identified, is responsible for both the cytostatic and cytotoxic effect. Clay et al., Biochemistry 34:11142-51 (1995). When tested against primary neurons in culture, these peptides were also found to elicit degeneration of neurites.
Purified apoE, derived from transfected HEK cells, subjected to thrombin cleavage and separated using gel filtration to collect the 22 kD fragment, yields enhanced toxicity when tested against primary neurons in culture. The 22 kD fragments purified from the E4 isoform is more toxic than E3derived fragments. The putative toxic site is closely associated with one of two well-characterized heparin binding domains associated with residues 141-147 of apoE.
The density of four positively charged amino acid residues in the 141-149 domain clearly make a significant contribution to apoE peptide toxicity. Consistent with this conclusion is the ablation of peptide-mediated toxicity by the polyanionic glycosaminoglycans ("GAG") heparin, heparin sulfate and chondroitin sulfate. However, GAG-binding capacity does not, in itself, account for bioactivity since peptide E211-243, which contains a second heparin-binding site but lacks the 141-149 sequence, is inactive. Furthermore, not all GAGs show inhibition of the toxicity.
While there has been considerable research into the mechanisms underlying Alzheimer's disease, there continues to be an ongoing need for new ways to investigate and combat this disorder and other diseases in which ApoE toxicity has been implicated.
U.S. Pat. No. 4,727,063 discloses low molecular weight heparins having a sulfation degree of at least 2.5 and a molecular weight ranging from 2000 to 9000, prepared by depolymerization and sulfation with a mixture of sulfuric and chlorosulfonic acid. None has a sulfation degree up to 3.5.
U.S. Pat. No. 3,454,560 discloses a process for the depolymerization and sulfabon of chondroitin sulfate by means of sulfuric acid at a concentration not lower than 85% w/w. The sulfuric acid can contain another sulfating agent, such as sulfuric anhydride or chlorosulfonic acid, but the same document specifies that, even operating in said ambient, only sulfuric acid participates in the sulfation reaction.
U.S. Pat. No. 5,508,167, Roses et al., issued Apr. 16, 1996, discloses methods of diagnosing or prognosing Alzheimer's disease in a subject. The methods involve directly or indirectly detecting the presence or absence of an apolipoprotein E type (ApoE4) isoform or DNA, encoding ApoE4 in the subject. The presence of ApoE4 indicates the subject is afflicted with Alzheimer's disease or at risk of developing Alzheimer's disease. A novel immunochemical assay for detecting the presence or absence of the Apolipoprotein E (ApoE) E4 allele in a subject is also disclosed.
U.S. Pat No. 5,384,398, Lormeau et al., issued Jan. 24, 1995, discloses new high molecular mass N,O-sulphated heparosans consisting of chains or of a mixture of chains having a molecular mass of between 1.5.times.104 and 4.0.times.10 D, characterized by a repeating disaccharide structure.
U.S. Pat. No. 5,164,295, Kisilevsky et al., issued Nov. 17, 1992, discloses a method of identifying compounds which impair and/or prevent initiation and/or progression of amyloid deposition, such compounds being useful as therapeutics for treating amyloidosis and amyloid-related disorders.
U.S. Pat. No. 4,956,347, Ban et al., issued Sep. 11, 1990, relates to the use of ATEROID, a mixture of "sulfomucopolysaccharides" comprising heparin, heparan sulfate-like substance, dermatan sulfate, and chondroitin sulfate A and C, for the treatment of patients suffering from Alzheimer's-type senile dementia. ATEROID is defined in the U.S. Pat No. 3,000,787, Bianchini, issued Sep. 19, 1961, as a heparinoid anti-cholesterolemic factor. ATEROID, which is in some aspects similar to heparin, has essentially no anti-coagulant effect The patent discloses that ATEROID can be extracted from the small intestine and particularly from the duodenum of mammals, by means of methods suitable for the isolation of aminopolysaccharidic or glycoproteic compounds.
Snow, A. D., et al., American Journal of Pathology, Vol. 133, No. Dec. 3, 1988, disclose the presence of heparan sulfate proteoglycan (HSPG) in neuritic plaques associated with Alzheimer's disease. HSPG was detected in the amyloid fibrils present in neuritic plaques in the brains of Alzheimer's patients using antibodies against the protein core of HSPG. Additionally, HSPG was shown to be present in primitive plaques. It is suggested that the accumulation of HSPG in plaques takes place during early stages of plaque development.
Snow, A. D., and Kisilevsky, R., Laboratory Investigation, Vol. 53, No. 1, pp. 37-44 (1985), report the temporal relationship between glycosaminoglycan (GAG) accumulation and amyloid deposition during experimental amyloidosis. Using models which facilitate induction of amyloidosis, it was disclosed that amyloid-associated GAGs appear in the tissues together with the AA amyloid protein independent of the tissue type. It is suggested that the appearance of GAG in the inflammatory amyloidosis condition appears to be part of the process involved in the deposition of the AA protein.
Margolis, R. U., and Margolis, R. K., Neurobiology of Aging, Vol. 10, pp. 500-502 (1989) disclose various properties of nervous tissue proteoglycans with respect to their proposed relation to amyloid beta-protein in Alzheimer's disease-related amyloidosis. It is pointed out on page 501, column 1, lines 4 to 8 that the role of proteoglycans in Alzheimer's disease amyloidosis is only circumstantial and the role of proteoglycans in it is unclear. At page 502, column 2, lines 3 to 6, it is disclosed that due to the absence of firm evidence specifically linking proteoglycans to pathogenesis of Alzheimer's disease, it is premature to speculate the relationship of proteoglycans to amyloid in degenerative process.
Caputo, C. B., Neurobiology of Aging, Vol. 10, pp. 503-504 (1989) refers to the significance of binding of proteoglycans to amyloid. It is disclosed that co-localization of proteoglycans with amyloids indicates that they are binding but the consequence of such binding is unknown. The question is asked of whether proteoglycans bind inadvertently to amyloid or whether the proteoglycans in binding to amyloid or its precursors lead to the formation of beta-pleated sheet conformation or the stabilization of such a conformation. It is suggested that in vitro studies be performed to determine whether Alzheimer amyloid precursor binds to proteoglycans. On page 503, column 1, last paragraph, the possibility that amyloid protein binds well to proteoglycans is raised. However, evidence is referred to which indicates otherwise.